Extract from the leaves of ginkgo biloba

ABSTRACT

An extract from Ginkgo biloba is prepared by separating a lipid fraction, mixing with an alcohol, extracting the alcoholic solution with an organic solvent to remove neutral lipids, and the alcoholic solution is dissolved in a solvent and applied to a chromatographic column. A number of extracting media are applied to the chromatographic column to extract various fractions. The particular fractions of interest for incorporation into topical compositions have characteristics of cerebrosides or digalactosyldiglycerides.

The invention relates to a new extract from the leaves of Ginkgo bilobaand its method of preparation.

Certain extracts from the leaves of Ginkgo biloba are widely used forthe therapy of peripheral and cerebral arterial circulatorydisturbances. Various processes for preparing such extracts aredescribed for example in DE-B -1767098, DE-B2117429, EP-A 0 324 197,EP-A 330 567 and EP-A 0 436 129.

The invention also provides a method for preparing a glycolipid extractfrom Ginkgo biloba leaves comprising the steps of:

extracting the leaves with an organic solvent;

separating the extract from the leaves;

cooling the extract to precipitate a lipid fraction;

recovering the lipid fraction from the extract;

mixing the lipid fraction with an alcohol;

removing insoluble material;

extracting the alcoholic solution with an organic solvent to removeneutral lipids;

treating the concentrated alcoholic solution with a number of extractingmedia;

purifying the extracting medium containing a desired fraction; and

substantially removing the associated extracting medium from the desiredfraction to provide a concentrated extract.

The process of the invention is a highly efficient extraction processfor extracting desired extracts, such as cerebrosides and especiallydigalactosyldiglycerides from the leaves of Ginkgo biloba. Theseextracts have applications in topical compositions.

In general terms in the process of the invention; the leaves of Ginkgobiloba are first extracted with an organic solvent such as acetone/waterand the resulting solution is concentrated, for example, by evaporationand cooled. The solution is then filtered and a C₁ -C₃ alcohol such asethanol is added to the precipitate. The solution is agitated and againfiltered. The filtrate is adjusted with water and extracted with asuitable organic solvent especially a non polar solvent such as heptane.The particular advantage of extraction with a non polar solvent prior toapplication to a chromatographic column is that the solvent removes asubstantial proportion, typically 30%, of the dry extract. As a resultthe amount of desired extract, particularly digalactosyldiglycerides, isincreased. The aqueous alcoholic phase is then evaporated to dryness, orevaporated and filtered and an organic solvent is added. The resultingsolution is filtered, and then purified by chromatographic separationusing solvent extraction media.

Preferably the organic solvent in which the concentrated alcoholicsolution in dissolved is the same as a first extracting medium appliedto the column. This optimises the chromatographic process.

In a preferred embodiment of the invention the concentrated alcoholicsolution from which neutral lipids have been removed is treated with atleast two different extracting media. The advantage of this feature isin facilitating the targeted extraction of a desired fraction from thematerial applied to the column.

Preferably at least one of the extracting media is an organic solvent.

In a preferred embodiment of the invention the organic solvent is amixture of acetone and toluene. We have found that this solvent isparticularly advantageous in targeting the extracts of interest. Thestrength of the mixture depends on the extract being targeted.Preferably the organic solvent in 5% to 80% acetone/toluene. In one casethe organic solvent is 20% to 80% acetone toluene, most preferably 40%to 50% acetone/toluene.

In another case where ginkgolic acids, which are generally undesirable,are to be extracted the acetone/toluene is preferably 5 to 30%,especially approximately 20%.

In another embodiment of the invention one of the extracting media withwhich the concentrated alcoholic solution from which neutral lipids havebeen removed is treated is a mixture of an organic solvent and analcohol. Preferably the alcohol is a C₁ to C₃ alcohol, especiallyethanol. Preferably also in this case the solvent is acetone. Theextracting medium may be 5% to 85% ethanol/acetone depending on thetarget extract. In one case it is 8% ethanol/acetone and in another 80%ethanol/acetone.

One of the extracting media may be acetone which is again a usefulsolvent for target extracts.

In a preferred embodiment of the invention the alcoholic solution isconcentrated prior to treatment with the extracting medium/media.Typically the alcoholic solution is concentrated by evaporation.

In one embodiment of the invention a desired fraction obtained byextraction is concentrated, redissolved and reprocessed through thechromatographic column to produce a more concentrated desired fraction.Preferably the medium containing the desired fraction is purified bytreating with charcoal and filtering the desired extract to remove thecharcoal.

In one preferred embodiment of the invention the concentrated alcoholicsolution is dissolved in 40% acetone toluene, applied to achromatographic column and 20% acetone/toluene, 40% acetone/toluene,acetone and 8% ethanol/acetone were applied sequentially to the column.In this case preferably the fraction extracted by the acetone wasconcentrated, redissolved in 40% acetone/toluene and reprocessed throughthe column. One of the extracts in this case has characteristics ofdigalactosyldiglycerides and another has characteristics ofcerebrosides.

In another preferred embodiment of the invention the concentratedalcoholic solution is dissolved in 80% acetone/toluene, applied to achromatographic column and 80% acetone/toluene, acetone, and 80% ethanolacetone were applied sequentially to the column. In this case preferablythe fraction extracted by the acetone was concentrated, washed anddried. The extract in this case has characteristics ofdigalactosyldiglycerides.

In a further preferred embodiment of the invention after treating thealcoholic solution with an organic solvent to remove neutral lipids thealcoholic solution is concentrated and filtered. In this case preferablythe precipitate is dissolved in 50% acetone/toluene, applied to achromatographic column, and 50% acetone/toluene and 8% ethanol/acetoneare applied sequentially to the column. Preferably the ethanol/acetonefraction is concentrated, redissolved in 70% acetone/toluene andreprocessed through the chromatographic column. The extract in this camehas characteristics of digalactosyldiglycerides at particularly highlevels of purity, greater than 80% and in some cases approximately 95%.

The invention also provides a topical composition including an extractof the invention.

The invention will be described in further detail with reference to thefollowing examples.

EXAMPLE 1

Step 1

60 kg of finely milled Ginkgo biloba leaves were extracted with 500 l ofa 60% acetone water mixture in a counter current extraction unit at atemperature of 55°-60° C.

The extract was separated from the leaves and was then concentrated byevaporation.

Step 2

The concentrate was cooled to 8°-10° C. and was retained at thistemperature for 3 hours.

The lipids which precipitated were recovered by decanting.

The recovered lipids were then dissolved in an agitated solution of 2.9kg of acetone and 0.7 kg of demineralised water. A further 30 kg ofdemineralised water was then added.

The solution was continuously agitated and cooled to 8°-10° C. and wasretained for a minimum of 3 hours before being decanted.

Step 3

The wet decanted lipids from Step 2 were added to 80 kg of ethanol andagitated for 3 hours.

Insoluble material was removed by decanting.

Step 4

The ethanolic solution from Step 3 was adjusted to 12-15% water.

The solution was extracted with 60 l of Heptane.

The ethanol-water phase was evaporated to greater than 85% dry extract.

Step 5

The alcoholic solution containing the extract from Step 4 was dissolvedin 40% acetone/toluene and retained at ambient temperature overnight.

This solution was filtered and was then applied to 18 kg of silica in apacked column. The following solvents were pumped through the column:-200 l 20% acetone/toluene, 400 l 40% acetone/toluene, 100 l acetone and120 l 8% ethanol/acetone.

Step 6

The desired fraction extracted by the third extraction medium (acetone)applied to the column was evaporated and redissolved in 40%acetone/toluene and reprocessed through the column using the sameconditions as above and three main fractions taken.

Step 7

Charcoal (1 part) was added to the desired acetone solution (10 partsDry Extract) and the solution was agitated for 30 minutes and filtered.The resulting solution was evaporated giving 120 g of a green brownsolid. This fraction was characterised using HPLC chromatography and amass detection for identification.

The extract from which substantially all of the neutral lipids andginkgolic acids were removed with the first (20% acetone/toluene)extracting solvent was analysed by HPLC analysis and exhibited threemajor peaks.

The first peak was characteristic of mono-galactosyldiglycerides. Thethird peak was characteristic of digalactosyldiglycerides which are thepreferred extract. The peaks eluting between the mono anddigalactosyldiglycerides may be cerebroside in character.

The second of the three fractions obtained in Step 6 above containsgreater than 70 to 80% of the material corresponding to the second majorpeak. This fraction may be cerebroside in character.

EXAMPLE 2

Step 1

35 kg of finely milled Ginkgo biloba leaves were extracted with 280 l ofa 60% acetone water mixture in a counter current extraction unit atemperature of 55°-60° C.

The extract was separated from the leaves and was then concentrated byevaporation.

Step 2

The concentrate was cooled to 8°-10° C. and was retained at thistemperature for 3 hours.

The lipids which precipitated were recovered by decanting.

The recovered lipids were then dissolved in an agitated solution of 1.4kg of acetone and 1.0 kg of demineralised water. A further 18 kg ofdemineralised water was then added.

The solution was continuously agitated and cooled to 8°-10° C. and wasretained for a minimum of 3 hours before being decanted.

Step 3

The wet decanted lipids from Step 2 were added to 34 kg of ethanol andagitated for 3 hours.

Insoluble material was removed by decanting.

Step 4

The ethanolic solution from Step 3 was adjusted to 12-15% water.

The solution was extracted with 40 l of Heptane.

The ethanol-water phase was evaporated to greater than 85% dry extract.

Step 5

The extract from Step 4 was dissolved in 80% acetone/toluene andretained at ambient temperature overnight.

This solution was filtered and was then applied to 18 kg of silica in apacked column. The following solvents were pumped through the column:-140 l 80% acetone/toluene, 130 l acetone and 120 l 80% ethanol/acetone.

Step 6

The acetone fraction was evaporated and the resulting material washedseveral times with acetone. The semi solid was dried to give a paleyellow solid which was identified and quantified on HPLC analysis asdigalactosyldiglycerides (45 g) at a purity of 95%.

The glyceride moiety of the digalactosyldiglycerides was identified byhydrolysis and gas chromatography analysis of the methyl esters.

C16:0 15.3%

C16:1 0.4%

C18:0 2.9%

C18:2 8.0%

C18:3 55.7%

Others 17.7%

EXAMPLE 3

Step 1

60 kg of finely milled Ginkgo biloba leaves were extracted with 500 l ofa 60% acetone water mixture in a counter current extraction unit at atemperature of 55°-60° C.

The extract was separated from the leaves and was then concentrated byevaporation.

Step 2

The concentrate was cooled to 8°-10° C. and was retained at thistemperature for 3 hours.

The lipids which precipitated were recovered by decanting.

The recovered lipids were then dissolved in an agitated solution of 2.9kg of acetone and 0.7 kg of demineralised water. A further 30 kg ofdemineralised water was then added.

The solution was continuously agitated and cooled to 8°-10° C. and wasretained for a minimum of 3 hours before being decanted.

Step 3

The wet decanted lipids from Step 2 were added to 80 kg of ethanol andagitated for 3 hours.

Insoluble material was removed by decanting.

Step 4

The ethanolic solution from Step 3 was adjusted to 12-15% water.

The solution was extracted with 60 l of Heptane.

The ethanol was evaporated and the resulting solution filtered.

Step 5

The precipitate from Step 4 was dissolved in 50% acetone/toluene andretained at ambient temperature overnight.

This solution was filtered and was then applied to 18 kg of silica in apacked column. The following solvent was pumped through the column:- 180l 50% acetone/toluene, and 120 l 8% ethanol/acetone.

Step 6

The ethanol/acetone fraction was evaporated and redissolved in 70%acetone/toluene and reprocessed through the column.

Step 7

The desired fraction rich in digalactosyldiglycerides was evaporated todryness to give a light brown solid. Precipitation with acetone yieldeddigalactosyldiglyceride.

The digalactosyldiglyceride and cerebroside extracts have shown activityin cosmetic and skin care applications.

The invention is not limited to the specific embodiments hereinbeforedescribed, which may be varied in detail.

I claim:
 1. A method for preparing an extract from Ginkgo biloba leavescomprising the steps of:extracting the leaves with a first organicsolvent; separating the extract from the leaves; cooling the extract toprecipitate a lipid fraction; recovering the lipid fraction from theextract; mixing the lipid fraction with an alcohol; removing insolublematerial; extracting the alcoholic solution with a second organicsolvent to remove neutral lipids and concentrate the alcoholic solution;treating the concentrated alcoholic solution with one or more ofextracting media; purifying the extracting media containing a desiredfraction; and substantially removing the associated extracting mediafrom the desired fraction to provide a concentrated extract.
 2. Themethod of claim 1 wherein the second organic solvent is a non polarsolvent.
 3. The method of claim 2 wherein the non polar solvent isheptane.
 4. The method of claim 1 wherein the concentrated alcoholicsolution is dissolved in a third organic solvent and the solution andthe extracting media are passed through a chromatographic column.
 5. Themethod of claim 4 wherein the third organic solvent is the same as thefirst extracting medium passed to the column.
 6. The method of claim 1wherein the alcoholic solution from which neutral lipids have beenremoved is treated with at least two different extracting media.
 7. Themethod of claim 6 wherein at least one of the extracting media is anorganic solvent.
 8. The method of claim 7 wherein the organic solvent isa mixture of acetone and toluene.
 9. The method of claim 8 wherein theorganic solvent contains 5% to 80% acetone/toluene.
 10. The method ofclaim 9 wherein the organic solvent contains from 20% to 80%acetone/toluene.
 11. The method of claim 10 wherein the solvent contains5% to 50% acetone/toluene.
 12. The method of claim 6 wherein theconcentrated alcoholic solution from which neutral lipids have beenremoved is treated with an extraction media which is a mixture of anorganic solvent and an alcohol.
 13. The method of claim 12 wherein thealcohol is a C₁ to C₃ alcohol.
 14. The method of claim 13 wherein thealcohol is ethanol.
 15. The method of claim 12 wherein the organicsolvent is acetone.
 16. The method of claim 12 wherein the extractingmedium is a 5% to 85% ethanol/acetone mixture.
 17. The method of claim16 wherein one of the extracting media is an 8% to 80% ethanol/acetonemixture.
 18. The method of claim 4 wherein one of the extracting mediais acetone.
 19. The method of claim 1 wherein the desired fraction issubstantially removed by precipitation with an organic solvent.
 20. Themethod of claim 1 wherein the alcoholic solution is concentrated priorto treatment with the extracting media.
 21. The method of claim 20wherein the alcoholic solution is concentrated by evaporation.
 22. Themethod of claim 1 wherein the concentrated extract is redissolved andreprocessed through the chromatographic column to further concentratethe desired fraction.
 23. The method of claim 22 wherein theconcentrated extract is purified by treating with charcoal and filteringto remove the charcoal.
 24. The method of claim 4 wherein theconcentrated alcoholic solution is dissolved in a 40% acetone/toluenemixture, passed through a chromatographic column and, thereafter, a 20%acetone/toluene mixture, a 40% acetone/toluene mixture, acetone and an8% ethanol/acetone mixture are passed sequentially through the column.25. The method of claim 22 wherein the fraction extracted by the acetoneis concentrated and redissolved in a 40% acetone/toluene mixture andreprocessed through the chromatographic column.
 26. The method of claim17 wherein the reprocessed extract has the characteristics ofdigalactosyldiglycerides.
 27. The method of claim 25 wherein thereprocessed extract has the characteristics of cerebrosides.
 28. Themethod of claim 4 wherein the concentrated alcoholic solution isdissolved in an 80% acetone/toluene mixture, passed through achromatographic column and, thereafter, an 80% acetone/toluene mixture,acetone, and an 80% ethanol/acetone mixture are sequentially passedthrough the column.
 29. The method of claim 28 wherein the fractionextracted by the acetone is concentrated, washed and dried.
 30. Themethod of claim 29 wherein the fraction extracted by acetone has thecharacteristics of digalactosyldiglycerides.
 31. The method of claim 1wherein, after treating the alcoholic solution with the second organicsolvent the concentrated alcoholic solution is filtered.
 32. The methodof claim 31 wherein the filtered precipitate is dissolved in a 50%acetone/toluene mixture, passed through a chromatographic column, and,thereafter, a 50% acetone/toluene mixture and an 8% ethanol/acetonemixture are sequentially passed through the column.
 33. The method ofclaim 32 wherein the ethanol/acetone fraction is concentrated,redissolved in a 70% acetone/toluene mixture and reprocessed through thechromatographic column.
 34. The method of claim 33 wherein the desiredfraction has the characteristics of digalactosyldiglycerides.
 35. Anextract from Ginkgo biloba leaves prepared by the method of claim
 1. 36.The extract of claim 35 which has characteristics ofdigalactosyldiglycerides.
 37. The extract of claim 35 having a purity ofgreater than 80%.
 38. The extract of claim 36 having a purity ofapproximately 95%.
 39. The extract claim 35 which has characteristics ofcerebrosides.
 40. A topical composition comprising an extract preparedby the method of claim
 1. 41. The method of claim 6 wherein one of theextracting media is acetone.
 42. The method of claim 24 wherein theconcentrated alcoholic solution is dissolved in an 80% acetone/toluenemixture, passed through a chromatographic column and, thereafter, an 80%acetone/toluene mixture, acetone, and an 80% ethanol/acetone mixture aresequentially passed through the column.
 43. The method of claim 25wherein the fraction extracted by the acetone is concentrated, washedand dried.
 44. The method of claim 26 wherein the fraction extracted byacetone has the characteristics of digalactosyldiglycerides.
 45. Acomposition comprising an extract predominantly comprising at least oneof monogalactosyldiglycerides, cerebrosides and digalactosyldiglyceridesprepared by the method of claim
 1. 46. A composition according to claim45 wherein the extract contains cerebrosides having a purity of at least70% to 80%.
 47. A composition according to claim 45 wherein the extractcontains digalactosyldiglycerides having a purity of at least 80%.
 48. Acomposition according to claim 45 wherein the extract containsdigalactosyldiglycerides having a purity of at least 95%.
 49. Acomposition according to claim 45 wherein the digalactosyldiglycerideshas a glyceride moiety selected from C16:0, C16:1, C18:0, C18:2 andC18:3.